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Mikamo Lecture
Signaling Circuitry in Heart Failure: Lessons from Mice to Men
Richard A. Walsh, M.D.
Case Western Reserve University
University Hospitals of Cleveland
Cleveland, Ohio
  • Changing Concepts
  • Mechanisms for Compensated Hypertrophy
  • Protein Kinase C
  • PKC Activation and Heart Failure
  • Mechanisms for PKC Activation
  • PKC in Pathologic Hypertrophy
  • Molecular Therapeutic Targets


  • Signal transduction pathways are the molecular cascades responsible for communicating information from the exterior to the interior of the cell. Abnormalities in these pathways play a pivotal role in the production of heart failure.





    Changing Concepts


    The biochemical paradigm recognizing that the activation of the neurohormonal pathways, in particular the renin angiotensin and sympathetic nervous systems, may importantly contribute to the evolution of heart failure has supplanted the traditional hemodynamic paradigm of heart failure. This biochemical paradigm serves as a basis for current cardiovascular therapeutics in heart failure. The understanding in the last decade that abnormalities in myocardial and vascular cell types causally contribute to heart failure has provided for new therapeutic opportunities. Understanding molecular and cell biology and how they contribute to the onset of heart failure will serve as a platform for new and effective therapeutic modalities.

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    Mechanisms for Compensated Hypertrophy


    Figure 1. Overload-induced hypertrophy results in a concentric increase in cardiac contractile units.
    Click to enlarge
    Hypertrophy, an increase in cardiac contractile units in a concentric fashion as a consequence of pressure overload or volume overload is illustrated in Figure 1. The increase in contractile units normalizes wall stress, maintains left ventricular systolic function and maintains cardiac output. The pressure or volume overload, if sufficiently intense or prolonged, results in a large, dilated heart with an increase in wall stress and a mechanical disadvantage for both pumping and filling.

    Abnormalities in signal transduction and how they may contribute to or cause maladaptive hypertrophy and heart failure is the focus of much research. Some of the signal transduction pathways identified as being important in the production of pathologic hypertrophy and heart failure, include cytokines, conventional growth factors, angiotensin, extracellular stresses and mechanical deformation. For example, if GP130 in the JAK-STAT pathway, which has been shown to be anti-apoptotic or protective for normal growth, is knocked out and superimposed with pressure overload, dilated cardiomyopathy occurs in the genetically-engineered mouse on the basis of massive apoptosis.

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    Protein Kinase C


    The protein kinase C (PKC) pathway seems to occupy a nodal position and can cross-activate the classical MAP kinase pathway. In other cell types it has been shown to be important in the production of pathologic growth and abnormalities of function.

    Angiotensin II, endothelin-1, phenylephrine, prostaglandin-F2-alpha and mechanical deformation can activate protein kinase C. All are involved in the pathologic development of pressure overload, volume overload and heart failure. The coupling of angiotensin and the other hormones to their cognate receptor activates, by virtue of dissociation of the Gqalpha symbol subunit of the heterotrimeric G-protein, phospholipase C-beta-1 (PLC-ß1). PLC-ß1 acts upon phosphitol inositol bisphosphate to produce diacylglycerol (DAG), a general stimulant of PKC, and inositol triphosphate, a modulator of intracellular calcium homeostasis.

    Certain PKC isoforms are calcium-dependent, thus there is a positive feedback loop for PKC activation of conventional isoforms. This calcium release may, in incompletely defined ways, stimulate the activation of phosphatases and other calcium-dependent kinases.

    The different isoforms of PKC include the serine threonine kinases, initially described as a pathway activated by partial lipid hydrolysis. The classical protein kinases, represented by alpha, beta-1, beta-2 and gamma, have a calcium-binding domain and can be activated by diacylglycerol, phorbol ester, phosphatidylserine or calcium. The so-called novel protein kinases, represented by delta and epsilon, lack the calcium-binding domain and can be activated independently of calcium by phosphatidylserine and diacylglycerol. The atypical protein kinases family lacks the diacylglycerol PMA binding domain and can be activated by phosphatidylserine, independent of calcium or diacylglycerol.

    The existence of different isoforms of PKC has suggested that they may have different functions, and this fundamental question is of great research interest. Walsh and colleagues, using pressure overload in a guinea pig model, showed that four weeks of descending thoracic aortic banding was associated with an increase in left ventricular (LV) mass, which was associated with normal isovolumic LV contractile function and normal isolated cardiomyocyte mechanics. But, after 8 weeks of banding a decrease in isovolumic LV performance and cardiomyocyte function was observed. Thus, this served as a convenient reagent to study molecular events common to either the compensated or decompensated pressure overload hypertrophy and heart failure.

    They also demonstrated that acute mechanical stretch to a minimum diastolic pressure of 25 mm Hg was sufficient to activate phospholipase C with inositol triphosphate accumulation, PKC translocation and activation, mediated in part by angiotensin II. Further, PKC promiscuous activation by a phorbol ester depresses isolated LV mechanics, suggesting that hyperactivation of the PKC pathway might contribute to contractile dysfunction in heart failure.

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    PKC Activation and Heart Failure


    In an experiment to assess whether and to what extent LV performance seen as a clinical consequence of ACE inhibition was due to blockade of PKC activation, they showed there was no change in the components of the PKC signal transduction pathway. But, there was hyperactivation of PKC isoforms and elevated PKC activity with chronic pressure overload and heart failure in this guinea pig model. The differential PKC activation was mediated by increases in Gaq and PLC-ß1 hydrolysis, rather than up-regulation of their expression.

    They have also shown that survival in response to LV pressure overload by descending thoracic aortic banding was enhanced by ACE inhibition in this guinea pig model of decompensated congestive heart failure. They demonstrated that LV hypertrophy and heart failure was diminished and that LV and cardiomyocyte function was improved, indicating improvement in LV performance was partly intrinsic. There was upregulation of the calcium cycling proteins and an increase in the calcium transient associated with a decrease in PKC translocation. PKC has been shown in vitro, by other laboratories, to downregulate the calcium cycling proteins shown to be dysfunctional and decreased their abundance in heart failure.

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    Mechanisms for PKC Activation


    Mechanical deformation, G-protein coupled receptor activation by angiotensin and endothelin, has been shown to activate PKC. Walsh and colleagues have shown that angiotensin II, hypoxia and ischemia activate a variety of the PKC isoforms via phospholipase-C and tyrosine kinase coupled pathways. Interestingly, however, oxidative stress using hydrogen peroxide was able to activate PKC independently of PLC-ß1 or tyrosine kinase signal transduction—which has important therapeutic implications.

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    PKC in Pathologic Hypertrophy


    Walsh and colleagues have used genetically-engineered mice to understand and characterize the phenotypic consequences of the genetic modifications in pathologic hypertrophy. Their work sought to answer these questions: Is the phenotype due to altered expression of the transgene itself? Are similar alterations in protein abundance found in experimental animal systems or human disease? Is the observed phenotype a consequence of an insertional effect of the transgene? Is there ectopic expression of the transgene in a location it usually does not reside? What are the consequences of the poison peptide, the nonspecific overexpression in large non-physiologic, non-pathologic ways of a protein? How much of the phenotype is due to phenotypic changes?

    Walsh and colleagues showed that overexpression of PKC-beta-2 in the transgenic mouse produces ventricular hypertrophy, myocardial fibrosis, and depression of LV function. Further, they showed that isoform inhibition, using the PKC-ß2 inhibitor, prevented or regressed pathologic hypertrophy and normalized LV function. In terms of the molecular mechanism for the dysfunction due to overexpression of PKC-beta-2, they showed a decrease in the rate of shortening and the rate of re-lengthening, associated with normal calcium transients. This suggests that the abnormal in vivo function was, at least in part, due to a decrease in calcium sensitivity at the level of the cardiomyocyte. Superfusion with the PKC inhibitor resulted in relative normalization of isolated LV cardiomyocyte mechanics.

    They concluded that PKC-beta-2 overexpression depressed myocyte shortening and re-lengthening and is associated with normal intracellular calcium transients. Using a survey of phosphorylation targets in the cardiomyocyte, they found increased phosphorylation of troponin-I, which resulted in the decrease in myofilament calcium sensitivity. The PKC-ß2 inhibitor improved cardiomyocyte function. PKC inhibition might prove to be a useful therapy for human heart failure.

    In another study they showed that PKC isoform expression and activity are increased in the failing human heart due to increased protein expression of beta-1, beta-2, and alpha (calcium-dependent isoforms). There was a non-significant reduction in the abundance and translocation of PKC-epsilon. The increased isoform expression was at least in part due to increases in activity in the cardiomyocyte that were transcriptionally mediated. PKC activity was reduced by 45% with the PKC-b2 inhibitor, again suggesting the importance of PKC activation of classical isoforms in cardiomyopathic congestive heart failure.

    Figure 2. Transgenic Expression of PKC Isoforms.
    Click to enlarge

    Figure 3. Conceptual framework of the transition from cardiomyocyte hypertrophy to heart failure.
    Click to enlarge
    Walsh and colleagues then went on to show that targeted transgenic overexpression of the PKC-epsilon isoform produces concentric LV hypertrophy with no cardiac fibrosis. In vivo LV function was normal with markedly depressed calcium transients, suggesting an enhancement of calcium sensitivity of the myofilament. There were no alterations in the abundance and phosphorylation status of the calcium cycling proteins. Figure 2 summarizes the differences between the two isoforms.

    In summary, PKC activation in cardiac hypertrophy and heart failure has been demonstrated at the level of neonatal cardiomyocytes in conventional models of heart failure. Genetically-engineered mice with overexpression in an isoform-specific manner of PKC-beta can produce cardiac hypertrophy and failure, whereas PKC-epsilon produces a normal form of cardiac hypertrophy.

    An elegant study that interfered with Gqalpha symbol function showed that LV hypertrophy in response to banding was substantially prevented. Isoform-specific abnormalities of PKC occur in human heart failure. PKC is activated by diverse pathologic stimuli, including stress, ischemia, hypoxia and reactive oxygen species, all of which play a role in the development of heart failure. Figure 3 illustrates the overall conceptual framework. Angiotensin, endothelin and phenylephrine signal through the PKC pathway. Oxidative stress and stretch may directly activate PKC. These stimuli, acting in concert, can produce cardiomyocyte hypertrophy, pathologic hypertrophy and contractile depression and heart failure.

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    Molecular Therapeutic Targets


    Potential therapeutic targets in heart failure include GPCR inhibition, which acts in part via a decrease in PKC activation. This is being done with ACE inhibition and AT-1 receptor blockade. Gqalpha symbol inhibition has been done in the genetically-engineered mouse, but is not likely to be used clinically because of the beneficial effects of some of the PKC isoforms. Isoform-specific PKC inhibition, for example with PKC-beta, is an attractive therapeutic target. Activation of the extrinsic off switches in a generic way might be possible. RACK inhibition of the pathologic PKC isoform activation is a particularly attractive target. RACKs (receptors for activated C kinases) are molecules that act as intracellular receptors or docking sites for PKC and may define or at least contribute importantly to PKC isoform specificity. Isoform-specific PKC activation, by contrast, using a similar strategy, may be possible for PKC-epsilon. Among the subcellular deleterious targets for enhanced oxidative stress, which is known to occur in heart failure, is activation of PKC.

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